Alpha-synuclein (?Syn) pathology is linked to synucleinopathies including Parkinson's disease and Lewy body dementia, but the underlying disease mechanisms remain poorly understood. The prevalent viewpoint has emerged that aggregation of ?Syn triggers neuropathology through a gain-of-toxic-function mechanism, and approaches to eliminate ?Syn represent an active area of research for treatment. Yet, ?Syn aggregation may also endanger neurons by removing ?Syn from synaptic vesicles (its physiologically relevant intracellular location) and thereby causing loss-of-function. Through its synaptic vesicle-bound state, ?Syn regulates synaptic vesicle trafficking, and chaperones SNARE-complex assembly to maintain neurotransmitter release. Thus, removing ?Syn from neurons may not be protective, but detrimental. The objective in this application is to determine the impact of synaptic vesicle-binding of ?Syn on ?Syn function and neuron survival, using rationally designed variants of ?Syn that stabilize synaptic vesicle-binding. The central hypothesis is that stabilizing binding of ?Syn on synaptic vesicles reduces ?Syn toxicity and pathology. Guided by strong preliminary data, this hypothesis will be tested in three specific aims: 1) Determine the effect of increased synaptic vesicle-binding of ?Syn on SNARE-complex assembly; 2) Assess the effect of increased synaptic vesicle-binding of ?Syn on synaptic vesicle cycling; and 3) Test if increased synaptic vesicle-binding of ?Syn rescues neurotoxicity and pathology in vivo. Under the first aim, SNARE-complex assembly will be quantified in vivo and in vitro, using cell biological and biochemical techniques. Under the second aim, ?Syn multimerization, synaptic vesicle pools and clustering, and synaptic vesicle cycling will be quantified, using cell biological, biochemical and biophysical techniques. Under the third aim, mouse models will be generated by stereotactic injections of lentiviral vectors into the substantia nigra of ?Syn knockout mice to assess effects of mutant ?Syn variants on ?Syn-induced toxicity and pathology, using behavioral assays on mice and biochemical, histological and ultrastructural analyses on injected brains. The study is expected to show improved ?Syn function and delayed pathology upon stabilization of synaptic vesicle-binding of ?Syn. This research is innovative because it 1) tests the novel hypothesis that stabilizing synaptic vesicle-bound ?Syn reduces ?Syn pathology, 2) creates new tools to study function and dysfunction of ?Syn, and 3) uses a multidisciplinary approach to test our hypothesis from single molecules and cellular systems to live mice. This work is significant, because it will 1) clarify the importance of synaptic vesicle-binding of ?Syn for neuron function, 2) provide new insights into the molecular mechanism of synaptic vesicle-binding of ?Syn, 3) uncover the contribution of loss-of-function of ?Syn to disease pathogenesis, and 4) have translational importance for the development of new treatment strategies aimed at stabilizing synaptic vesicle-bound ?Syn.